Abstract

The RNA exosome is a key 3'-5' exoribonuclease with an evolutionarily conserved structure and function. Its cytosolic functions require the co-factors SKI7 and the Ski complex. Here we demonstrate by co-purification experiments that the ARM-repeat protein RESURRECTION1 (RST1) and RST1 INTERACTING PROTEIN (RIPR) connect the cytosolic Arabidopsis RNA exosome to the Ski complex. rst1 and ripr mutants accumulate RNA quality control siRNAs (rqc-siRNAs) produced by the post-transcriptional gene silencing (PTGS) machinery when mRNA degradation is compromised. The small RNA populations observed in rst1 and ripr mutants are also detected in mutants lacking the RRP45B/CER7 core exosome subunit. Thus, molecular and genetic evidence supports a physical and functional link between RST1, RIPR and the RNA exosome. Our data reveal the existence of additional cytosolic exosome co-factors besides the known Ski subunits. RST1 is not restricted to plants, as homologues with a similar domain architecture but unknown function exist in animals, including humans.

The wax-deficient phenotype of rst1 mutants is caused by silencing of the CER3 gene. a Inflorescence stems of Arabidopsis plants of the indicated genotypes. GFP-RST1 and RST1-GFP are rst1-3 plants expressing RST1 fused to GFP. The whitish appearance of wild-type stems is due to a layer of the epicuticular wax deposited on the stem surface, while wax-deficient stems appear green and glossy. To better visualise the difference between normal and glossy stems, the white balance of the photograph was set to cold light (3800 K), which accounts for the bluish appearance of the picture. b Relative amounts of major stem wax compounds extracted from Arabidopsis stem sections. c Levels of CER3 mRNA and CER3-derived siRNAs in WT, cer7 and rst1 mutants. The total RNA extracted from stem samples of the indicated genotypes was separated by denaturing agarose (left) or polyacrylamide (right) electrophoresis, transferred to membranes and hybridised with a probe specific to CER3. The methylene blue (MB) stained membrane and hybridisation with a probe specific to U6 snRNA are shown as loading controls, respectively. d Mutating SGS3 restores the wax phenotype of rst1 mutants. Stem sections from rst1-2 and rst1-2 sgs3-13 plants are shown on the left. RNA blots show full-length CER3 mRNA (mid), and CER3-derived siRNAs (right) in RNA samples extracted from stems of the indicated genotypes. The source data are available at [10.6084/m9.figshare.c.4483406]

RST1 suppresses silencing of transgenes. a The rst1-4 mutation suppresses the developmental phenotype induced by a MIM156 transgene. b Diagram of the AT3G27670 gene encoding the RST1 protein. Boxes represent exons, lines represent introns. Triangles indicate the position of the T-DNA insertions in rst1-2 and rst1-3 lines. Vertical lines indicate the point mutations in rst1-4 and rst1-5. crst1-4 is a weak allele. Accumulation of the CER3 mRNA (left) and CER3-derived siRNAs (right) in wild-type (WT), cer7-3 and the four rst1 alleles used in this study shown by RNA blots hybridised with a probe specific to CER3. The methylene blue stain of the membrane (MB) and hybridisation to U6 snRNA are shown as loading controls. d RNA blots showing the accumulation of the full-length MIM156 ncRNA and MIM156-derived siRNAs visualised by hybridisation with a probe specific to the IPS1 backbone of the MIM156 transgene. 7SL RNA and U6 snRNA are shown as loading controls. e RNA blots showing that the rst1-5 mutation results in reduced levels of the GUS mRNA and increased levels of GUS-derived siRNAs in the L1 jmj14-4 background. 25S rRNA and U6 snRNA are shown as loading controls. f The rst1-5 mutation increases S-PTGS frequency in both 6b4 and Hc1 reporter lines. The barplot shows the proportion of plants with silenced GUS expression in the indicated genotypes. The source data are available at [10.6084/m9.figshare.c.4483406]

RST1 is a cytosolic protein. Confocal microscopy of root tips from Arabidopsis rst1-3 plants expressing GFP-RST1 and RST1-GFP fusion proteins under the control of the constitutive UBIQUITIN 10 promoter. Poly(A)-binding protein 2 at its N-terminus fused to tRFP (tRFP-PAB2) and expressed under the control of its own promoter is shown as cytosolic marker. Scale bars are 10 µm. The source data are available at [10.6084/m9.figshare.c.4483406]

RST1 co-purifies with the exosome, SKI7 and RIPR. Volcano plots show the enrichment of proteins co-purified with GFP-tagged RST1 (a) or RRP41 (b) as compared with control IPs. Y- and X-axis display adjusted p-values and fold changes, respectively. The dashed line indicates the threshold above which proteins are significantly enriched (adjP < 0.05). The source data are available in Supplementary Data and

RST1 and RIPR are bound to CER7-containing exosomes. Volcano plots show the enrichment of proteins co-purified with GFP-tagged RRP45B/CER7 (a) or RRP45A (b) as compared with control IPs. Y- and X-axis display adjusted p-value and fold change, respectively. The dashed line indicates the threshold above which proteins are significantly enriched (adjP< 0.05). The source data are available in Supplementary Data

RIPR co-immunoprecipitates RST1, SKI7 and the Ski complex. The Volcano plot shows the enrichment of proteins co-purified with GFP-tagged RIPR as compared with control IPs. Y- and X-axis display adjusted p-value and fold change, respectively. The dashed line indicates the threshold above which proteins are significantly enriched. The source data are available in Supplementary Data

Loss of RIPR function phenocopies rst1 mutants. a Confocal microscopy of plants expressing RIPR-GFP, the cytosolic marker tRFP-PAB2 and a GFP-tagged version of the exosome subunit RRP4. Scale bars are 20 µm. The source data are available at [10.6084/m9.figshare.c.4483406]. b Scheme of the AT5G44150 gene encoding the RIPR protein. Small and large boxes represent exons in the UTRs and CDS, respectively, lines represent introns. The single insertion of a T or C nucleotide at position 179 (from the ATG) results in a frameshift, which creates a premature stop codon. c Electropherograms of the genomic DNA sequence surrounding the relevant position of the AT5G44150 locus in wild-type, ripr(insT) and ripr(insC) plants. d Stem sections from wild-type (WT), cer7, rst1 and ripr plants. ecer7-3, rst1 and ripr mutants produce similar proportions of non-viable seeds. Scale bar is 0.5 mm. Please also see the uncropped pictures provided in Supplementary Fig. . f Northern blots showing the downregulation of the CER3 mRNA (left) and the upregulation of CER3-derived siRNAs (right) in ripr mutants. Loading controls show the methylene blue stained membrane (MB, left) and the hybridisation with a probe specific to U6 snRNA (right). The uncropped blots can be seen in Supplementary Fig. , and are also available at [10.6084/m9.figshare.c.4483406]. g RIPR is a silencing suppressor. The barplot shows the percentage of plants that spontaneously trigger silencing of the Hc1 35S prom:GUS S-PTGS reporter

Loss of RST1 or RIPR results in the accumulation of small RNAs that are also produced in cer7 mutants a Multidimensional scaling plot illustrating global variance and similarities between the 21/22 nt small RNA populations detected in the replicates of WT, cer7, rst1 and ripr. b Venn diagram showing that rst1 and ripr mutants accumulate quasi identical populations of small RNAs almost all of which are also detected in cer7 mutants. The source data are available in Supplementary Data